Epithelial cells present in mesenchymal organs (e.g., blood, bone marrow or lymph nodes) can be detected by the expression and/or release of epithelial-specific cytoskeleton proteins called cytokeratins. For example, epithelial tumor cells derived from carcinomas such as breast, prostate, lung or colon cancers may be detected at the single cell level using cytokeratins (Pantel K, Brakenhoff R H, 2004, Nat Rev Cancer 4:448-56; Alix-Panabières C, et al., 2007, Clin Chem 53:537-9).
Cytokeratins constitute the largest intermediate filament protein subgroup and represent a multigene family with more than 20 different types of polypeptides that are divided into relatively acidic type I (CK9-CK20) and basic type II (CK1-CK8) keratins (Moll R, et al., 1982, Cell, 31:11-24.). Cells of normal epithelium express at least one type I and one type II keratin. Cytokeratins form the cytoskeleton of epithelial cells and their main function is to maintain the epithelial cell integrity. The expression of cytokeratins varies with the type of epithelial cells, and usually is maintained in epithelial tumor cells (e.g., breast, colon, prostate cancer, etc.) (Steinert et al., 1988, Annu Rev Biochem 57:593-625; Chu et al., 2002, Histopathology 40:403-39) although changes may occur in the expression pattern of individual cytoskeletal proteins in human breast carcinoma cells (Willipinski et al., 2005, Clin Cancer Res 11:8006-8014.). Extra-cellular cytokeratins are detected either as partially degraded single protein fragments, as small complexes, or as large polymeric protein complexes (Rydlander et al., 1996, Eur J Biochem 241:309-14). Multiple mechanisms including abnormal mitosis, spill over of monomeric cytokeratin polypeptides from dividing cells, proteolytic degradation of cytokeratin in apoptotic cells, and/or neovascularisation has been suggested. In addition, extra-cellular cytokeratin fragments may be found circulating in the blood due to infection.
CK19, one of the three main keratins with CK8 and CK18, is found in simple or stratified epithelium and in carcinomas, and has been demonstrated to be stably and abundantly expressed in primary epithelial tumors such as breast, colon, lung and hepatocellular cancer cells, but not in mesenchymal hematopoietic cells. Several studies have shown that upon cell stress induced by apoptosis, type I cytokeratins become substrates for caspase 3 proteases and specifically the proteolysis of full length CK19 by caspase 3 to produce the CK19 soluble fragment, CYFRA 21-1, which is released in supernatant of cultured cells, and in the serum of lung cancer patients (Fuchs et al., 1994, Annu Rev Biochem 63:345-82; Coulombe, P A, 1993, Curr Opin Cell Biol 5:17-29; Ku N O et al., 1997, J Biol Chem 272:33197-203; Dohmoto et al., 2001, Int J Cancer 91:468-473; Sheard et al., 2002, J Cell Biochem 85:670-677). Moreover, Ding et al., (2004, Mol Cell Proteomics 3:73-81) demonstrated that overexpression of intracellular CK19 in hepatocellular carcinoma is related to metastatic behavior. However, the release of the full length (intact) CK19 protein has not been reported.
CYFRA 21-1 measurement in the circulation has been established for monitoring patients with lung cancers and in head and neck cancers (Bodenmuller et al., 1994, Int J Biol Markers 9:75-81; Nisman et al., 1998, Cancer 82:1850-9; Pujol et al., 1993, Cancer Res 53:61-6). Intra-cellular CK19 has been also used as a marker for the detection of micrometastatic cancer cells in the bone marrow, lymph nodes, and peripheral blood by immunocytochemistry (ICC) and RT-PCR (Braun et al., 2000, N Engl J Med 2000. 342:525-533; Schoenfeld et al., 1997, Eur J Cancer 33:854-861). However, these methods are not able to distinguish between viable and apoptotic cells, and they are not suitable to detect secreted marker proteins.
A need exists for a method of detecting viable epithelial cells that release a full-length cytokeratin. A method of detecting a released, full-length cytokeratin as a marker of disseminated epithelial cells present in a non-epithelial sample is also needed to indicate the presence of tumor cells.